Plant biotechnology has the potential to improve human health on a number of fronts. Plants can be engineered to produce therapeutic proteins as drugs. Crops can be genetically modified to produce food that is more nutritious and to require less chemical fertilizers and pesticides. Plants can also be engineered to clean up the environment or replace the fossil fuels that threaten the atmosphere. But one critical problem faced by plant biotechnology is the inability to produce large amounts of transgenic proteins in plant cells. This limitation is particularly challenging in the realm of drug production where the amount of protein produced by a plant influences the economic viability of the drug. One way to address the problem of low protein production in plants is to find new places in the plant cell to store engineered proteins. The goal of this proposal is to discover and characterize new compartments in the plant cell for accumulation of transgenic proteins. Currently, targeting proteins to the cytosol and membrane bound organelles like the endoplasmic reticulum results in only low concentrations of the protein per gram of plant tissue. This reduces the profitability of plant biotechnology when compared to other strategies like protein expression in bacteria and mammalian cell cultures. If successful, this project will identify new organelles or subcellular structures that will improve the efficiency of plant genetic engineering. The project will also determine how to target proteins to these new locations. In addition, this project may identify new biochemical pathways as targets for future plant genetic engineering. This project will begin by looking for new organelles in the model plant Arabidopsis thaliana. A set of 108 unique transgenic Arabidopsis have been produced that express different fusions between the green fluorescent protein (GFP) and random plant proteins (1). These random proteins serve as targeting signals to send the GFP to different intracellular compartments. In some cases, the GFP has been found to accumulate in regions of the cell never before observed by plant biologists. This project will aim to characterize these new organelles and how proteins can be targeted to them. The initial screening stage of this project will use fluoresce microscopy to identify candidate plants in which GFP lights up new structures in the plant cell. The transgenes will then be cloned out of the candidate lines to determine what protein sequences are targeting the GFP to their particular locations. This sequence information will be used to determine the minimal requirements for targeting proteins to the new organelle. Finally, to fully characterize the organelles, they will be purified and analyzed for protein content by peptide fingerprinting.